PEG / Ion. User Guide HR2-139 (pg 1) glycol 3,350. Refer to the enclosed PEG/Ion HT reagent formulation for additional information on all 96 reagents.

Transcription

1 PEG / Ion TM User Guide HR2-139 (pg 1) Applications Crystallization screen for soluble biological macromolecules. Features Reagent formulation developed at Hampton Research Screens a profile of anions, cations, multivalent ions, titrated organic acids at varying ph levels in the presence of monodisperse Polyethylene glycol 3,350 Tacsimate ph 4, 5, 6, 7, 8, and Polyethylene glycol ph range Novel CBTP buffer component Tryptone (peptide library) General Description PEG/Ion HT is a crystallization reagent kit designed to provide a rapid screening method for the crystallization of biological macromolecules. PEG/Ion HT is designed as a 96 reagent crystallization screen that combines the strategies of PEG/Ion Screen (HR2-126) and PEG/Ion 2 Screen (HR2-098) into a highly effective and efficient format. This kit allows one to evaluate a large variety of potential crystallization conditions with the 96 unique reagents. PEG/Ion HT is supplied in a sterile, polypropylene 96 Deep block, each reservoir containing 1 ml of sterile filtered reagent. The block is compatible with robotic and multi-channel pipet liquid handling systems and is heat sealed using a special polypropylene backed film. Each PEG/Ion HT kit is supplied with an adhesive clear sealing film (HR3-609) and one cap mat (HR3-103), which can be used to seal the block after removing the heat seal. Within the 96 Deep block, rows A through D feature the 48 reagents of PEG/Ion Screen (HR2-126). The screen combines high purity Polyethylene glycol 3,350 and 48 different high purity salts, comprising both anions (sulfate, nitrate, tartrate, acetate, chloride, iodide, thiocyanate, formate, citrate, phosphate, and fluoride) and cations (sodium, potassium, ammonium, lithium, magnesium, and calcium) in a relatively low concentration (0.2 M) which due to their unique ph characteristics also affords a reasonable ph screen (approximate ph range of 4 to 9). The primary screen variables are PEG, ion type, ionic strength, and ph. Rows E through H feature the 48 reagents of PEG/Ion 2 Screen (HR2-098). PEG/Ion 2 Screen is a crystallization reagent kit designed to provide a rapid screening method for the crystallization of biological macromolecules in the presence of Polyethylene glycol 3,350 and an array of neutralized and ph adjusted organic acids, multivalent ions, a novel Citrate BIS-TRIS propane buffer system (CBTP), Tryptone (peptide library), and ph. PEG/Ion 2 Screen utilizes a monodisperse (Mr 3,300-3,400), high purity, Polyethylene glycol 3,350. Refer to the enclosed PEG/Ion HT reagent formulation for additional information on all 96 reagents. Sample Preparation The macromolecular sample should be homogenous, as pure as is practically possible (>95%) and free of amorphous and particulate material. Remove amorphous material by centrifugation or micro-filtration prior to use (1, 2, 3). The recommended sample concentration is 5 to 25 mg/ml in sample buffer. Initially, the sample should be free of any unnecessary additives in order to observe the effect of the PEG/Ion HT variables. Ideally, the initial screen should be performed with a sample which has been dialyzed against dilute buffer although ligands, ions, reducing agents, or other additives may be present as required by the sample for solubility, stability, or activity. Preparing the Deep Block for Use It is recommended the Deep block be centrifuged and at 25 degrees Celsius before removing the sealing film. Centrifugation at 500 rpm for five minutes will remove stray reagent from the sealing film. Removing the reagent from the film prevents stray reagent droplets from falling into neighboring wells during film removal. After centrifugation the film can be removed by grasping a corner of the film and gently peeling the film from the plate. Alternatively, the film can be left intact and the pierced for reagent access. Performing The Screen Since it is the most frequently reported method of crystallization, the following procedure describes the use of the PEG/Ion HT with the Sitting Drop Vapor Diffusion method. The PEG/Ion HT is also very compatible with the Hanging Drop, Sandwich Drop, Microbatch, and Microdialysis methods. A complete description of the Hanging, Sitting, Sandwich Drop, Dialysis and other crystallization methods are available from the Hampton Research Crystal Growth 101 Library. Manual Method - Sitting Drop Vapor Diffusion 1. Using a 96 well sitting drop vapor diffusion plate, pipet the recommended volume (typically 50 to 100 microliters) of crystallization reagent from the Deep block into the reservoirs of the crystallization plate. The Deep block is compatible with 8 and 12 channel pipets as well as many automated liquid handling systems. Use clean pipet tips for each reagent set transfer and change pipet tips when changing reagents. For an 8 channel pipet, transfer reagents A1-H1 to reservoirs A1-H1 of the crystallization plate. Repeat this procedure for reagent columns B through H. Change pipet tips

2 PEG / Ion TM User Guide HR2-139 (pg 2) when moving between reagent columns. For a 12 channel pipet, transfer reagents A1-A12 to reservoirs A1-A12 of the crystallization plate. Repeat this procedure for reagent rows 1 through 12. See Figure 1 below. Time and pipet tips can be conserved by batch pipetting multiple plates with the same (row or column) of reagent before changing reagent and pipet tips. Figure 3 Figure 1 2. Using clean pipet tips, pipet 0.05 to 2 microliters of crystallization reagent from the crystallization plate reservoir to the sitting drop well. Some 96 well crystallization plates allow this procedure to be performed using a multichannel pipet where other plates require the use of a single channel pipet. Change the pipet tip between reagents. See Figure 2 below. 2. nce the plate is oiled, use an 8 or 12 channel pipet to aspirate reagent from the Deep block and dispense the reagent under the oil in the Microbatch plate. Change tips when changing reagent to prevent cross reagent contamination. To save time and pipet tips, set multiple plates at one time. See Figure 4. Figure 4 Figure 2 3. Using a single channel pipet, aspirate the sample and dispense the sample under oil in the Microbatch plate. It is not necessary to dispense the sample drop into the reagent drop or mix the drops. See Figure Using a clean pipet tip, pipet 0.05 to 2 microliters of sample to the reagent drop in the sitting drop well. ne may choose to simply dispense the sample with no mixing or dispense with mixing by gently aspirating and dispensing the sample several times, keeping the tip in the drop during mixing to avoid foaming. Work carefully but quickly to minimize evaporation from the crystallization plate. See Figure Seal the crystallization plate as per the manufacturers recommendation. Most 96 well crystallization plates are sealed using a clear sealing tape or film. View and score the experiment as desired. See Hampton Research technical bulletin Crystal Growth Viewing Crystallization Experiments for additional information on viewing drops. Manual Method Microbatch 96 Format 1. Using a 96 well clear polystyrene microplate (U-bottom recommended for best drop centering, flat-bottom recommended for best optics) pipet approximately 150 microliters of Microbatch compatible oil into each of the 96 reservoirs. This can be accomplished using an 8 or 12 channel pipet and pipetting the oil from a reagent basin. See Figure 3. Figure 5 4. After all reagent and sample drops have been dispensed to the Microbatch plate, place the loose fitting clear cover on the Microbatch plate and centrifuge the plate for 10 minutes at 500 rpm. Centrifugation will cause the drops to coalesce into a single drop. Note: If the drops appear flat or is fragmented into multiple drops, the centrifugation speed is too high and the centrifugation time it too long adjust to obtain a spherical single drop in the center of the well. 5. Store the plates with the loose fitting clear polystyrene cover and observe for crystals. See Hampton Research technical bulletin Crystal Growth Viewing Crystallization Experiments for additional information on viewing drops.

3 PEG / Ion TM User Guide HR2-139 (pg 3) Figure 4 Typical observations in a crystallization experiment Clear Drop Skin / Precipitate Precipitate Precipitate / Phase Quasi Crystals Microcrystals Needle Cluster Plates Rod Cluster Single Crystal PEG/Ion HT Deep Block and Automated Liquid Handling Systems The polypropylene Deep block is designed to be compatible with the SBS standard 96 microwell format and is therefore compatible with numerous automated liquid handling systems that accept 8x12 96 well assay blocks. Follow the manufacturer s recommendation for handling deep well microplates. Examine the Drop Carefully examine the drops under a stereo microscope (10 to 100x magnification) immediately after setting up the screen. Record all observations and be particularly careful to scan the focal plane for small crystals. bserve the drops once each day for the first week, then once a week there after. Records should indicate whether the drop is clear, contains precipitate, and or crystals. It is helpful to describe the drop contents using descriptive terms. Adding magnitude is also helpful. Example: 4+ yellow/brown fine precipitate, 2+ small bipyramid crystals, clear drop, 3+ needle shaped crystals in 1+ white precipitate. ne may also employ a standard numerical scoring scheme (Clear = 0, Precipitate = 1, Crystal = 10, etc). Figure 4 (on page 3) shows typical examples of what one might observe in a crystallization experiment. Interpreting PEG/Ion HT Clear drops indicate that either the relative supersaturation of the sample and reagent is too low or the drop has not yet completed equilibration. If the drop remains clear after 3 to 4 weeks consider repeating the screen condition and doubling the sample concentration. If more than 70 of the 96 screen drops are clear consider doubling the sample concentration and repeating the entire screen. Drops containing precipitate indicate either the relative supersaturation of the sample and reagent is too high, the sample has denatured, or the sample is heterogeneous. To reduce the relative supersaturation, dilute the sample twofold and repeat the screen condition. If more than 70 of the 96 screen drops contain precipitate and no crystals are present, consider diluting the sample concentration in half and repeating the entire screen. If sample denaturation is suspect, take measures to stabilize the sample (add reducing agent, ligands, glycerol, salt, or other stabilizing agents). If the sample is impure, aggregated, or heterogeneous take measures to pursue homogeneity. It is possible to obtain crystals from precipitate so do not discard nor ignore a drop containing precipitate. If possible, examine drops containing precipitate under polarizing optics to differentiate precipitate from microcrystalline material. If the drop contains a macromolecular crystal the relative supersaturation of the sample and reagent is appropriate for crystal nucleation and growth. The next step is to optimize the preliminary conditions (ph, salt type, salt concentration, precipitant type, precipitant concentration, sample concentration, temperature, additives, and other crystallization variables) which produced the crystal in order to improve crystal size and quality. Compare the observations between the 4 C and room temperature incubation to determine the effect of temperature on sample solubility. Different results in the same drops at different temperatures indicate that sample solubility is temperature dependent and that one should include temperature as a variable in subsequent screens and optimization experiments. Retain and observe plates until the drops are dried out. Crystal growth can occur within 15 minutes or one year. PEG/Ion HT Formulation Crystallization reagents are formulated using the highest purity chemicals, ultrapure water (18.2 Megohm-cm, 5 ppb TC) and are sterile filtered using 0.22 micron filters into sterile Deep blocks (no preservatives added). PEG/Ion HT reagents are readily reproduced using Hampton Research ptimize and Stockptions stock solutions of salts, polymers and buffers. Refer to PEG/Ion Screen and PEG/Ion 2 Screen Fundamentals for further information regarding reagent formulation. ptimize and Stockptions stock reagents make reproducing crystallization screen reagents accurate, precise, fast, convenient and easy. Dilutions can be performed directly into the crystallization plate using ptimize and Stockptions stock reagents. PEG/Ion HT reagents are stable at room temperature and are best used before the Best If Used By. To enhance reagent stability it is strongly recommended that PEG/Ion HT reagents be stored at 4 C or -20 C. Avoid ultraviolet light to preserve reagent stability. If the sample contains phosphate, borate, or carbonate buffers

4 PEG / Ion TM User Guide HR2-139 (pg 4) it is possible to obtain inorganic crystals (false positives) when using crystallization reagents containing divalent cations such as magnesium, calcium, or zinc. To avoid false positives use phosphate, borate, or carbonate buffers at concentrations of 10 mm or less or exchange the phosphate, borate, or carbonate buffer with a more soluble buffer that does not complex with divalent cations. References and Readings 1. Crystallization of nucleic acids and proteins, Edited by A. Ducruix and R. Giege, The Practical Approach Series, xford Univ. Press, Current approaches to macromolecular crystallization. McPherson, A. Eur. J. Biochem. 189, 1-23, Protein and Nucleic Acid Crystallization. Methods, A Companion to Methods in Enzymology, Academic Press, Volume 1, Number 1, August Technical Support Inquiries regarding PEG/Ion HT reagent formulation, interpretation of screen results, optimization strategies and general inquiries regarding crystallization are welcome. Please , fax, or telephone your request to Hampton Research. Fax and Technical Support are available 24 hours a day. Telephone technical support is available 8:00 a.m. to 5:00 p.m. USA Pacific Standard Time. Hampton Research 34 Journey Aliso Viejo, CA U.S.A. Tel: (949) Fax: (949) Technical Support tech@hrmail.com Website: Hampton Research Corp. all rights reserved Printed in the United States of America. This guide or parts thereof may not be reproduced in any form without the written permission of the publishers.

5 PEG / Ion HT HR2-139 Reagent Formulation 1. (A1) 2. (A2) 3. (A3) 4. (A4) 5. (A5) 6. (A6) 7. (A7) 8. (A8) 9. (A9) 10. (A10) 11. (A11) 12. (A12) 13. (B1) 14. (B2) 15. (B3) 16. (B4) 17. (B5) 18. (B6) 19. (B7) 20. (B8) 21. (B9) 22. (B10) 23. (B11) 24. (B12) 25. (C1) 26. (C2) 27. (C3) 28. (C4) 29. (C5) 30. (C6) 31. (C7) 32. (C8) 33. (C9) 34. (C10) 35. (C11) 36. (C12) 37. (D1) 38. (D2) 39. (D3) 40. (D4) 41. (D5) 42. (D6) 43. (D7) 44. (D8) 45. (D9) 46. (D10) 47. (D11) 48. (D12) Salt 0.2 M Sodium fl uoride 0.2 M Potassium fl uoride 0.2 M Ammonium fl uoride 0.2 M Lithium chloride 0.2 M Magnesium chloride hexahydrate 0.2 M Sodium chloride 0.2 M Calcium chloride dihydrate 0.2 M Potassium chloride 0.2 M Ammonium chloride 0.2 M Sodium iodide 0.2 M Potassium iodide 0.2 M Ammonium iodide 0.2 M Sodium thiocyanate 0.2 M Potassium thiocyanate 0.2 M Lithium nitrate 0.2 M Magnesium nitrate hexahydrate 0.2 M Sodium nitrate 0.2 M Potassium nitrate 0.2 M Ammonium nitrate 0.2 M Magnesium formate dihydrate 0.2 M Sodium formate 0.2 M Potassium formate 0.2 M Ammonium formate 0.2 M Lithium acetate dihydrate 0.2 M Magnesium acetate tetrahydrate 0.2 M Zinc acetate dihydrate 0.2 M Sodium acetate trihydrate 0.2 M Calcium acetate hydrate 0.2 M Potassium acetate 0.2 M Ammonium acetate 0.2 M Lithium sulfate monohydrate 0.2 M Magnesium sulfate heptahydrate 0.2 M Sodium sulfate decahydrate 0.2 M Potassium sulfate 0.2 M Ammonium sulfate 0.2 M Sodium tartrate dibasic dihydrate 0.2 M Potassium sodium tartrate tetrahydrate 0.2 M Ammonium tartrate dibasic 0.2 M Sodium phosphate monobasic monohydrate 0.2 M Sodium phosphate dibasic dihydrate 0.2 M Potassium phosphate monobasic 0.2 M Potassium phosphate dibasic 0.2 M Ammonium phosphate monobasic 0.2 M Ammonium phosphate dibasic 0.2 M Lithium citrate tribasic tetrahydrate 0.2 M Sodium citrate tribasic dihydrate 0.2 M Potassium citrate tribasic monohydrate 0.2 M Ammonium citrate dibasic 1. (A1) 2. (A2) 3. (A3) 4. (A4) 5. (A5) 6. (A6) 7. (A7) 8. (A8) 9. (A9) 10. (A10) 11. (A11) 12. (A12) 13. (B1) 14. (B2) 15. (B3) 16. (B4) 17. (B5) 18. (B6) 19. (B7) 20. (B8) 21. (B9) 22. (B10) 23. (B11) 24. (B12) 25. (C1) 26. (C2) 27. (C3) 28. (C4) 29. (C5) 30. (C6) 31. (C7) 32. (C8) 33. (C9) 34. (C10) 35. (C11) 36. (C12) 37. (D1) 38. (D2) 39. (D3) 40. (D4) 41. (D5) 42. (D6) 43. (D7) 44. (D8) 45. (D9) 46. (D10) 47. (D11) 48. (D12) Polymer 1. (A1) 2. (A2) 3. (A3) 4. (A4) 5. (A5) 6. (A6) 7. (A7) 8. (A8) 9. (A9) 10. (A10) 11. (A11) 12. (A12) 13. (B1) 14. (B2) 15. (B3) 16. (B4) 17. (B5) 18. (B6) 19. (B7) 20. (B8) 21. (B9) 22. (B10) 23. (B11) 24. (B12) 25. (C1) 26. (C2) 27. (C3) 28. (C4) 29. (C5) 30. (C6) 31. (C7) 32. (C8) 33. (C9) 34. (C10) 35. (C11) 36. (C12) 37. (D1) 38. (D2) 39. (D3) 40. (D4) 41. (D5) 42. (D6) 43. (D7) 44. (D8) 45. (D9) 46. (D10) 47. (D11) 48. (D12) ph F - Fluoride N Cl - Chloride - - S C Thiocyanate Nitrate N I - Iodide C C - CH3 - H 3 Acetate Formate - P - - S Phosphate Sulfate H H - C C C C - H H Tartrate - H C H - C C C C C - H H H Citrate Measured ph at 25 C PEG/Ion HT contains ninety-six unique reagents. To determine the formulation of each reagent, simply read across the page.

6 PEG / Ion HT 49. (E1) 50. (E2) 51. (E3) 52. (E4) 53. (E5) 54. (E6) 55. (E7) 56. (E8) 57. (E9) 58. (E10) 59. (E11) 60. (E12) 61. (F1) 62. (F2) 63. (F3) 64. (F4) 65. (F5) 66. (F6) 67. (F7) 68. (F8) 69. (F9) 70. (F10) 71. (F11) 72. (F12) 73. (G1) 74. (G2) 75. (G3) 76. (G4) 77. (G5) 78. (G6) 79. (G7) 80. (G8) 81. (G9) 82. (G10) 83. (G11) 84. (G12) 85. (H1) 86. (H2) 87. (H3) 88. (H4) 89. (H5) 90. (H6) 91. (H7) 92. (H8) 93. (H9) 94. (H10) 95. (H11) 96. (H12) Salt 0.1 M Sodium malonate ph M Sodium malonate ph M Sodium malonate ph M Sodium malonate ph M Sodium malonate ph M Sodium malonate ph M Sodium malonate ph 0.2 M Sodium malonate ph 4% v/v Tacsimate ph 4.0 8% v/v Tacsimate ph 4.0 4% v/v Tacsimate ph 5.0 8% v/v Tacsimate ph 5.0 4% v/v Tacsimate ph 6.0 8% v/v Tacsimate ph 6.0 4% v/v Tacsimate ph 8% v/v Tacsimate ph 4% v/v Tacsimate ph 8.0 8% v/v Tacsimate ph M Succinic acid ph 0.2 M Succinic acid ph 0.1 M Ammonium citrate tribasic ph 0.2 M Ammonium citrate tribasic ph 0.1 M DL-Malic acid ph 0.2 M DL-Malic acid ph 0.1 M Sodium acetate trihydrate ph 0.2 M Sodium acetate trihydrate ph 0.1 M Sodium formate ph 0.2 M Sodium formate ph 0.1 M Ammonium tartrate dibasic ph 0.2 M Ammonium tartrate dibasic ph 2% v/v Tacsimate ph 4.0 2% v/v Tacsimate ph 5.0 2% v/v Tacsimate ph 6.0 2% v/v Tacsimate ph 2% v/v Tacsimate ph M Calcium chloride dihydrate, 0.02 M Cadmium chloride hydrate, 0.02 M Cobalt(II) chloride hexahydrate 0.01 M Magnesium chloride hexahydrate M Nickel(II) chloride hexahydrate 0.02 M Zinc chloride 0.15 M Cesium chloride 0.2 M Sodium bromide 1% w/v Tryptone 1% w/v Tryptone 49. (E1) 50. (E2) 51. (E3) 52. (E4) 53. (E5) 54. (E6) 55. (E7) 56. (E8) 57. (E9) 58. (E10) 59. (E11) 60. (E12) 61. (F1) 62. (F2) 63. (F3) 64. (F4) 65. (F5) 66. (F6) 67. (F7) 68. (F8) 69. (F9) 70. (F10) 71. (F11) 72. (F12) 73. (G1) 74. (G2) 75. (G3) 76. (G4) 77. (G5) 78. (G6) 79. (G7) 80. (G8) 81. (G9) 82. (G10) 83. (G11) 84. (G12) 85. (H1) 86. (H2) 87. (H3) 88. (H4) 89. (H5) 90. (H6) 91. (H7) 92. (H8) 93. (H9) 94. (H10) 95. (H11) 96. (H12) Buffer 0.1 M Sodium acetate trihydrate ph M Sodium citrate tribasic dihydrate ph M BIS-TRIS ph M HEPES ph M Tris ph M Citric acid, 0.03 M BIS-TRIS propane / ph M Citric acid, 0.04 M BIS-TRIS propane / ph M Citric acid, 0.05 M BIS-TRIS propane / ph M Citric acid, 0.06 M BIS-TRIS propane / ph M Citric acid, 0.07 M BIS-TRIS propane / ph M Citric acid, 0.08 M BIS-TRIS propane / ph M HEPES sodium ph 0.05 M HEPES sodium ph 0.05 M HEPES sodium ph HR2-139 Reagent Formulation 49. (E1) 50. (E2) 51. (E3) 52. (E4) 53. (E5) 54. (E6) 55. (E7) 56. (E8) 57. (E9) 58. (E10) 59. (E11) 60. (E12) 61. (F1) 62. (F2) 63. (F3) 64. (F4) 65. (F5) 66. (F6) 67. (F7) 68. (F8) 69. (F9) 70. (F10) 71. (F11) 72. (F12) 73. (G1) 74. (G2) 75. (G3) 76. (G4) 77. (G5) 78. (G6) 79. (G7) 80. (G8) 81. (G9) 82. (G10) 83. (G11) 84. (G12) 85. (H1) 86. (H2) 87. (H3) 88. (H4) 89. (H5) 90. (H6) 91. (H7) 92. (H8) 93. (H9) 94. (H10) 95. (H11) 96. (H12) Polymer 15% w/v Polyethylene glycol 3,350 15% w/v Polyethylene glycol 3,350 Buffer ph is that of a 1.0 M stock prior to dilution with other reagent components: ph with HCl or NaH. PEG/Ion HT contains ninety-six unique reagents. To determine the formulation of each reagent, simply read across the page.

7 Sample: Sample Buffer: Reservoir Volume: Sample Concentration: Date: Temperature: Drop Volume: Total μl Sample μl Reservoir μl Additive μl 1 Clear Drop 2 Phase Separation 3 Regular Granular Precipitate 4 Birefringent Precipitate or Microcrystals 5 Posettes or Spherulites 6 Needles (1D Growth) 7 Plates (2D Growth) 8 Single Crystals (3D Growth < 0.2 mm) 9 Single Crystals (3D Growth > 0.2 mm) PEG / Ion HT TM - HR2-139 Scoring Sheet Date: Date: Date: Date: 1. (A1) 0.2 M Sodium fl uoride, 2. (A2) 0.2 M Potassium fl uoride, 3. (A3) 0.2 M Ammonium fl uoride, 4. (A4) 0.2 M Lithium chloride, 5. (A5) 0.2 M Magnesium chloride hexahydrate, 6. (A6) 0.2 M Sodium chloride, 7. (A7) 0.2 M Calcium chloride dihydrate, 8. (A8) 0.2 M Potassium chloride, 9. (A9) 0.2 M Ammonium chloride, 10. (A10) 0.2 M Sodium iodide, 11. (A11) 0.2 M Potassium iodide, 12. (A12) 0.2 M Ammonium iodide, 13. (B1) 0.2 M Sodium thiocyanate, 14. (B2) 0.2 M Potassium thiocyanate, 15. (B3) 0.2 M Lithium nitrate, 16. (B4) 0.2 M Magnesium nitrate hexahydrate, 17. (B5) 0.2 M Sodium nitrate, 18. (B6) 0.2 M Potassium nitrate, 19. (B7) 0.2 M Ammonium nitrate, 20. (B8) 0.2 M Magnesium formate dihydrate, 21. (B9) 0.2 M Sodium formate, 22. (B10) 0.2 M Potassium formate, 23. (B11) 0.2 M Ammonium formate, 24. (B12) 0.2 M Lithium acetate dihydrate, 25. (C1) 0.2 M Magnesium acetate tetrahydrate, 26. (C2) 0.2 M Zinc acetate dihydrate, 27. (C3) 0.2 M Sodium acetate trihydrate, 28. (C4) 0.2 M Calcium acetate hydrate, 29. (C5) 0.2 M Potassium acetate, 30. (C6) 0.2 M Ammonium acetate, 31. (C7) 0.2 M Lithium sulfate monohydrate, 32. (C8) 0.2 M Magnesium sulfate heptahydrate, 33. (C9) 0.2 M Sodium sulfate decahydrate, 34. (C10) 0.2 M Potassium sulfate, 35. (C11) 0.2 M Ammonium sulfate, 36. (C12) 0.2 M Sodium tartrate dibasic dihydrate, 37. (D1) 0.2 M Potassium sodium tartrate tetrahydrate, 38. (D2) 0.2 M Ammonium tartrate dibasic, 39. (D3) 0.2 M Sodium phosphate monobasic monohydrate, 40. (D4) 0.2 M Sodium phosphate dibasic dihydrate, 41. (D5) 0.2 M Potassium phosphate monobasic, 42. (D6) 0.2 M Potassium phosphate dibasic, 43. (D7) 0.2 M Ammonium phosphate monobasic, 44. (D8) 0.2 M Ammonium phosphate dibasic, 45. (D9) 0.2 M Lithium citrate tribasic tetrahydrate, 46. (D10) 0.2 M Sodium citrate tribasic dihydrate, 47. (D11) 0.2 M Potassium citrate tribasic monohydrate, 48. (D12) 0.2 M Ammonium citrate dibasic, HR Page 1 of 2

8 Sample: Sample Buffer: Reservoir Volume: Sample Concentration: Date: Temperature: Drop Volume: Total μl Sample μl Reservoir μl Additive μl 1 Clear Drop 2 Phase Separation 3 Regular Granular Precipitate 4 Birefringent Precipitate or Microcrystals 5 Posettes or Spherulites 6 Needles (1D Growth) 7 Plates (2D Growth) 8 Single Crystals (3D Growth < 0.2 mm) 9 Single Crystals (3D Growth > 0.2 mm) PEG / Ion HT TM - HR2-139 Scoring Sheet Date: Date: Date: Date: 49. (E1) 0.1 M Sodium malonate ph 4.0, 50. (E2) 0.2 M Sodium malonate ph 4.0, 51. (E3) 0.1 M Sodium malonate ph 5.0, 52. (E4) 0.2 M Sodium malonate ph 5.0, 53. (E5) 0.1 M Sodium malonate ph 6.0, 54. (E6) 0.2 M Sodium malonate ph 6.0, 55. (E7) 0.1 M Sodium malonate ph, 56. (E8) 0.2 M Sodium malonate ph, 57. (E9) 4% v/v Tacsimate ph 4.0, 58. (E10) 8% v/v Tacsimate ph 4.0, 59. (E11) 4% v/v Tacsimate ph 5.0, 60. (E12) 8% v/v Tacsimate ph 5.0, 61. (F1) 4% v/v Tacsimate ph 6.0, 62. (F2) 8% v/v Tacsimate ph 6.0, 63. (F3) 4% v/v Tacsimate ph, 64. (F4) 8% v/v Tacsimate ph, 65. (F5) 4% v/v Tacsimate ph 8.0, 66. (F6) 8% v/v Tacsimate ph 8.0, 67. (F7) 0.1 M Succinic acid ph, 68. (F8) 0.2 M Succinic acid ph, 69. (F9) 0.1 M Ammonium citrate tribasic ph, 70. (F10) 0.2 M Ammonium citrate tribasic ph, 71. (F11) 0.1 M DL-Malic acid ph, 72. (F12) 0.2 M DL-Malic acid ph, 73. (G1) 0.1 M Sodium acetate trihydrate ph, 74. (G2) 0.2 M Sodium acetate trihydrate ph, 75. (G3) 0.1 M Sodium formate ph, 76. (G4) 0.2 M Sodium formate ph, 77. (G5) 0.1 M Ammonium tartrate dibasic ph, 78. (G6) 0.2 M Ammonium tartrate dibasic ph, 79. (G7) 0.1 M Sodium acetate trihydrate ph 4.6, 2% v/v Tacsimate ph 4.0, 80. (G8) 0.1 M Sodium citrate tribasic dihydrate ph 5.6, 2% v/v Tacsimate ph 5.0, 81. (G9) 0.1 M BIS-TRIS ph 6.5, 2% v/v Tacsimate ph 6.0, 82. (G10) 0.1 M HEPES ph 7.5, 2% v/v Tacsimate ph, 83. (G11) 0.1 M Tris ph 8.5, 2% v/v Tacsimate ph 8.0, 84. (G12) (0.07 M Citric acid, 0.03 M BIS-TRIS propane / ph 3.4), 85. (H1) (0.06 M Citric acid, 0.04 M BIS-TRIS propane / ph 4.1), 86. (H2) (0.05 M Citric acid, 0.05 M BIS-TRIS propane / ph 5.0), 87. (H3) (0.04 M Citric acid, 0.06 M BIS-TRIS propane / ph 6.4), 88. (H4) (0.03 M Citric acid, 0.07 M BIS-TRIS propane / ph 7.6), 89. (H5) (0.02 M Citric acid, 0.08 M BIS-TRIS propane / ph 8.8), 90. (H6) 0.02 M Calcium chloride dihydrate, 0.02 M Cadmium chloride hydrate, 0.02 M Cobalt(II) chloride hexahydrate, 91. (H7) 0.01 M Magnesium chloride hexahydrate, M Nickel(II) chloride hexahydrate 0.1 M HEPES sodium ph, 15% w/v Polyethylene glycol 3, (H8) 0.02 M Zinc chloride, 93. (H9) 0.15 M Cesium chloride, 15% w/v Polyethylene glycol 3, (H10) 0.2 M Sodium bromide, 95. (H11) 1% w/v Tryptone, 0.05 M HEPES sodium ph, 96. (H12) 1% w/v Tryptone, 0.05 M HEPES sodium ph, HR Page 2 of 2